A Handy Reagent for Testing the Reducing Power of Sugars Arvid Sandell Department of Inorganic Chemistry 1, Chemical Center, P. 0 . B. 124,s-221 00 Lund, Sweden I t is well known that certain kinds of sugar canin hot, alkaline solution reduce copper(I1)to more or less red copper(1) oxide. The reaction is complicated, and the stoichiometrv depends on the experimental conditions. Thus. the reaction ishardly more &an qualitative. With certain~recautions. however. is it ~ossibleto run the reaction a t least semi-quantitatively, If this reaction is to be used as a reagent for reducing sugar, it is necessary to keep the copper(I1) in solution by means of a suitable complex-formingligand. Otherwise the copper(I1) ions would precipitate in the alkaline medium as some bluish, sparingly soluble compound that we for simplicity's sake may call copper(I1) hydroxide, Cu(OH)2. Upon heating this precipitate would form brownish-black copper(I1)oxide, CuO, and totally mask any copper(1)oxide formed. The two procedures for this redox reaction, which are most frequently used for educational purposes, are perhaps those according to Trommer and Fehling, respectively. When a test according to Trommer is performed, no separate ligand is added. The test solution is made alkaline, whereupon a solution of coppedII) sulfate is added. With this method, it is necessary that the test solution contains a sufficient amount of a sugar, which in the alkaline solution forms complex with copper(I1)thus preventing precipitation of copper(I1) hydroxide. Otherwise the above-mentioned dark copper(11) oxide will be formed upon heating. It is clear that this method is of dubious value even in qualitative connection,because the test solution has to contain abundant suear. - . if the test should work at all. With the Fehling method tartrate ion is used as a ligand. In practice it is found chat the readv-mixed rearent solution does not keep well, because various solid phases are formed, copper(I1) is reduced by the tartrate ion, and the solution is sensitive to light ( I ) . That is why this reagent is prepared and stored as two separate solutions, Fehling I and Fehling 11. Immediately before use, equal portions of these solutions are mixed. This can involve practical difficulties in students' work, especially if small volumes are to be mixed, which many chemistry teachers know. If the mixing is made properly, the Fehling method can give a t least semi-quantitative results. However, the deep blue or violet color of the ready-mixed reagent is a disadvantage, especially when the test solution contains only a small amount of reducing substance. Then the pupils often describe the result as a "green solution" or something similar. Since Fehling in 1848 proposed his method (2),a good deal has happened in the chemistrv of com~lexes.One can expect that %e disadvantages with"~ehlin2smethod could be avoided bv chaneine the tartrate ion for a more suitable ligand. ~ i t goodre& g I have tried ethylenediaminetetraacetate, EDTA. This ligaud is easily accessible on the market and is probably to be found in every chemical laboratory. This ion is stable and shows no tendency to reduce copper(I1). Therefore, the reagent can be prepared and stored as a single solution. The ion forms a sufficiently strong complex with the copper(I1)ion to prevent precipitation of copper(I1) hydroxide. With copper(1)theEDTAion
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Journal of Chemical Education
forms a considerably weaker complex and cannot prevent precipitation of the s~arinelv - " soluble coo~er(1) . . oxide. A suitable general purpose reagent solution can be prepared by solving 8.3 g CuS04. 5H20and 18.6 g disodiumdihydrogen-EDTA dihydrate in 800 mL de-ionized or distilled water, whereupon 200 mL of a 2.5 M sodium hydroxide solution is added. The total concentrations in the resulting solution will be 33 mM of Cu2+,50 mM of EDTA, and 0.4 M of hydroxide ion. The reagent is weaklv blue colored. so the formation of even s m a ~ a m o u n tof s c u 2 0 is easy td observe. The readymixed rearrent can be kept for a lone time without being d e s t r o y e d y ~solution, which stoodfor six years unprotected against daylight in a rubber stopped bottle of uncolored glass worked as well as a freshly prepared solution. When the reagent is used, the test solution and the reagent solution are mixed in a test tube, and the mixture is kept in boiling heat for about 10 seconds. &
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Caution: Heating a test tube in an open flame always implies a risk for "bumnine". . ... and the alkaline solution can cause eye injury l f thmga go wrong I t 1s strongly recommended that the heatrng should be done in a hmhng waterbath.
The reagent has been tested with expected results with the carbohydrates that usually are used in education-glucose. fructose. saccharose. maltose. ladose. starch. and cellulose. The reaction studied here takes dace onlv in alkaline medium. Thus, one has to make the'heated iolution sufficiently alkaline. It is easy to show experimentally that the reactions run nicely in a phosphate buffer at pH = 12.2. Already a t pH = 10.3 (carbonate buffer), however, the result is unsatisfactory At this pH value the copper(1) oxide will darken swifilv to brownish black. and the sensitivitv for glucose is too ibw. The DHeiven to the reagent stock solution itself is a Dractical qiescon depending;pon, how one is going to u& the reagent. The recipe suggested here will give a reagent that has proved convenient in all normal applications, and the solution is cheap and simple to prepare. When testing for reducing sugar in a strongly acidic solution, as, for instance, after hydrolyzing cellulose in wncentrated sulfuric acid, one should, of course, begin by adding solid sodium hydroxide or sodium carbonate to neutralize the test solution. If the reagent is used in testing other substances than the above-mentioned, it can be necessary to increase the pH-value by adding one or two heads of solid NaOH to 5 mL of the hot solution. This applies, for instance, if one wants to indicate the reducing power of a formalin solution. The reagent has been in use since 1970 in Swedish school and university laboratories for following the enzymatic decomposition of starch into reducing sugars, and only positive experiences have been reported. Literature Cited
